Vaccination is currently the only method that can effectively impede the spread of influenza viruses among people. Traditional egg-based influenza vaccine technologies suffer from their incapability of massive and rapid production of vaccines against circulating influenza if a global outbreak occurs. Here, a new type of potent influenza vaccines is proposed based on the PI's previous works on protein surface display. The core of this new technology is to display a viral protein such as avian influenza H5N1 hemagglutinin (HA) on yeast surface in a manner such that these recombinant yeasts can serve as vaccines against influenza. Yeast is known of being able to express functional HA. The surface display of HA can significantly facilitate the recognition of antigens by host immune systems and mediate an immunoadjuvant effect with yeast cell membrane components. Although yeast has different glycosylation capability as compared to mammalian cells, this limited glycosylation may actually be a benefit to formulating a yeast vaccine. It has been reported that the glycosylation of natural influenza viruses in humans and in the standard vaccine actually interferes with induction of immunity by preventing access of antibodies to the HA surface. This benefit will be investigated thoroughly in this work. Alternatively, glycosylated yeast will be employed to construct HA surface-displayed yeast vaccines for evaluating the alteration of immunity of these vaccines. To enhance the efficacy and immunogenicity of the yeast vaccines, a dual-protein surface display technique will be adopted to codisplay the HA with CD154. The codisplay of HA with CD154 will further stimulate the maturation of antigen presenting cells and promote the adaptive immune response. Taken together, a hypothesis was developed that HA surface-displayed yeast vaccines can elicit strong and protective immunity against influenza. Furthermore, these potent vaccines can be enhanced by co-displaying HA with CD154. A recent animal vaccination study conducted in PI's lab supports this hypothesis. This project is proposed to further verify this hypothesis, three aims are proposed: Aim 1, Generate HA surface presented yeast influenza vaccines; Aim 2, Characterize both humoral and cell-mediated immune responses induced by yeast vaccines in animals (mice); Aim 3, Demonstrate the protection of mice from lethal avian influenza using yeast vaccines. The long-term goals of this project are to translate the animal studies into preclinical studies, determine the immunogenicity of these yeast vaccines in humans, and to augment this technology to develop other vaccines for treating many virus- infection related diseases such as AIDS and cancers. Unlike virus-based vaccines, yeast vaccines are safe for use in humans, and vaccine storage does not require refrigeration. Moreover, they can be massively and rapidly produced at a low cost. In addition, these vaccines do not require the passage of virus through eggs, offering a possibility for vaccine seed strain development that more closely matches the original wild virus and translating potentially into a better immunogenic and effective response.